For centuries, lactic acid bacterial cultures have been used in food production due to their ability to convert sugars by fermentation into preserving organic acids, predominantly lactic acid and various metabolic products associated with the development in the food product of a desirable taste and flavour. Some lactic acid bacteria produce hydrolytic enzymes including peptidases, proteases and lipolytic enzymes. The production of such enzymes contribute e.g. to flavour development in cheeses.
An interesting characteristic of certain lactic acid bacterial strains is their ability to produce antimicrobial compounds or bacteriocins having an inhibitory effect on closely related bacterial species. Certain lactic acid bacterial bacteriocins are applied in the food industry as preservatives.
However, for industrial production of a wide range of desired fermented food products such as all the well-known traditional dairy products including yoghurt, acidophilus milk, butter and cheeses; fermented vegetables; fermented meat products and animal feed a large range of lactic acid bacterial cultures, each of which are adapted to particular types of food products are required. Such cultures are presently being selected from naturally occurring strains of lactic acid bacteria on the basis of characteristics such as their ability to ferment sugars in the food product to be fermented, specific growth temperature requirements, production of desired flavouring compounds, the specific combination of which characteristics renders an individually selected culture useful for the production of a particular food product but normally less useful for production of others.
Obviously, this presently used procedure for developing useful lactic acid cultures by selection of naturally occurring strains is cumbersome and costly. Furthermore, it has proven difficult to provide starter culture strains which combine all the required characteristics at an optimal level. Presently, this problem is usually solved by the use of starter cultures comprising a multiplicity of selected lactic acid bacterial strains each having one or several of the characteristics desirable for a particular food product. The necessity to use such mixed cultures will of course also add to the costs in the manufacture of starter cultures.
Based on their traditional and long term application in food manufacturing and the fact that they are considered as non-pathogenic the lactic acid bacteria are generally recognized as safe food ingredients even if they are present in a fermented food product as live bacteria in a very high number.
Currently, it is widely recognized that a substantial industrial need exists to find economically and technically more feasible ways of developing starter cultures. It is obvious that gene technology may provide the means to meet this need. In the present context it is crucial that lactic acid bacteria for food starter cultures which are developed by introduction of desired genes by use of gene technology can still be recognized as safe for consumption. It is therefore essential that recombinant plasmids in order to be useful as cloning vectors in this development of lactic acid bacteria meet all the safety criteria as defined hereinbefore including the feature that such vectors only contains DNA originating from lactic acid bacteria including wild-type plasmids isolated from lactic acid bacteria. It is assumed that recombined lactic acid bacteria will still be recognized as safe for food production insofar they only contains DNA of lactic acid bacterial origin.
However, a precondition for the commercial exploitation of gene technology in the manufacturing of genetically recombined starter cultures is that such cultures would be generally recognized as safe for the consumers of food products containing live recombined lactic acid bacteria. One obvious potential risk associated with the ingestion of live recombined bacteria is the transfer of undesired genetic information herefrom to the indigenous gastro-intestinal microflora. Basically, this risk can be circumvented by constructing the recombined starter cultures in such a way that three conditions are fulfilled: (1) only DNA originating from lactic acid bacteria including wild-type plasmids isolated therefrom is introduced into the naturally occurring parent strains, (2) the inserted DNA is located on a cloning vector which essentially does not replicate in other bacterial species than lactic acid bacteria and (3) the inserted lactic acid bacterial DNA does not code for phenotypic traits which in the event they are conferred to the indigenous lactic acid bacterial flora could represent a health hazard.
The basis for the first condition is the fact that naturally occurring lactic acid cultures irrespective of their source are currently being considered as absolutely safe in food products. There has been no reports of any detrimental health effect by this traditional use of lactic acid bacteria. It is therefore generally considered that food starter cultures comprising recombined lactic acid bacterial DNA will be equally safe. The second and third conditions defined above, however, are based primarily on the consideration that mutants of naturally occurring strains of lactic acid bacteria may arise which thereby acquire undesired characteristics such as resistance to antibiotics which are used in pharmaceutical compositions useful for the treatment of infectious diseases. If such resistance phenotypes are transferred to infectious bacteria which may be present among non-lactic acid bacterial members of the human gastro-intestinal flora the treatment of such infections may become difficult. If lactic acid bacterial genes conferring resistance to pharmaceutically used antibiotics are inserted into a food starter culture there is a potential risk that such genes when the food starter-containing food is ingested might be transferred in-vivo to potentially pathogenic indigenous gastro-intestinal microorganisms.
The art of cloning foreign genes in lactic acid bacteria is still not well-developed. It is currently possible to construct recombinant strains hereof by transforming naturally occurring strains with recombinant plasmids into which desirable genes have been inserted and to obtain expression of such genes. However, the present state of the art does not provide the means of constructing recombinant starter cultures which fulfill the above-defined requirements of a safe recombined starter culture. In order to provide such safe starter cultures at least three major problems must be solved: (1) a suitable cloning vector must be provided which only contains DNA originating from a lactic acid bacterium including wild-type plasmids present herein, (2) the replication region of the cloning vector must be functional in lactic acid bacteria but preferably not in other bacterial species, in particular not in pathogenic or potentially pathogenic bacterial species, thereby reducing the risk of spreading of the vector to non lactic acid bacterial organisms to a minimum and (3) the cloning vector must be constructed in such a way that it contains a marker gene which is easily selectable in lactic acid bacteria and preferably not in other bacteria including pathogenic or potentially pathogenic gastro-intestinal bacteria, and which marker gene, should the highly unlikely event occur that the gene is transferred to a potentially harmful bacterium, would not add to the harmfulness of that bacterium e.g. by conferring resistance to a useful pharmaceutical antimicrobial agent.
Methods for the construction of cloning vectors which are functional in lactic acid bacteria are known, but vectors constructed by these known methods do not fulfill the requirements as defined above and are thus less acceptable as safe cloning means for food starter cultures.
As an example, EP 0 316 677 discloses a recombined vector which is constructed by joining a vector comprising a selectable marker which is preferably selected from genes conferring resistance to pharmaceutically applied antibiotics such as tetracycline, erythromycin and chloramphenicol and an origin of replication functional in an organism different from lactic acid bacteria such as E. coli, and a plasmid containing a replication region which is functional in lactic acid bacteria.
EP 0 228 726 discloses plasmid vectors which replicate in both gram-negative and gram-positive bacteria including lactic acid bacteria. Furthermore, the selectable markers which are used are preferably such antibiotic resistance traits which will allow selection in both gram-negative and gram-positive bacteria, a preferred selection marker being resistance to kanamycin.
A crucial step in the transformation of lactic acid bacteria with foreign DNA is the selection of transformants. Normally, the frequency of transformed cells in a transformation mixture is in the order of 10.sup.4 -10.sup.6 per .mu.g DNA, the frequency i.a. depending on the transformation method and the amounts of DNA and recipient cells in the transformation mixture. An effective selection of a relatively low number of transformed cells among non-transformed cells the number of which in a transformation mixture typically is in the range of 10.sup.8 -10.sup.10 requires an extremely effective selectable marker.
In the known methods of transformation of lactic acid bacteria the selectable markers inserted in cloning vectors are selected among genes conferring resistance to antibiotics. However, in any population of recipient cells in a transformation mixture, non-transformed cells occur which by spontaneous mutation have acquired resistance to the antibiotic to which the selectable marker confers resistance. The frequency of such spontaneous mutant cells may be of the same order as the above-defined frequency of transformation. In order to obtain reliable selection of transformants on a selective medium containing the antibiotic to which transformant cells have become resistant, the level of the antibiotic must be higher than that to which spontaneous mutants are resistant. The implication hereof is i.a. that the level of resistance conferred by the selectable marker must be correspondingly higher than the level of resistance acquired by spontaneous mutations.
In the present context another essential requirement for a suitable selectable marker is that the gene(s) coding for products conferring the resistance are expressed at a sufficiently high level immediately after transformation has occurred. If this is not the case, the selection procedure will become impractically long, maybe several days and the risk that spontaneous mutants overgrow the transformed cells will increase.
As defined above, a suitable selectable marker on a cloning vector for transformation of lactic acid bacterial food starter cultures should preferably be of lactic acid bacterial origin and furthermore, it should preferably not confer resistance to antibiotics which are also used as anti-infective agents in pharmaceutical compositions. Therefore, much interest has been concentrated on markers conferring resistance to a class of antibiotics which are produced naturally by certain strains of lactic acid bacteria, viz the so-called bacteriocins. Bacteriocins of lactic acid bacterial origin are not used in pharmaceutical compositions. Among such bacteriocins the polypeptide nisin produced by certain strains of Lactococcus spp is the best described.
Lactococcus spp producing nisin comprise at the same time genes mediating resistance to this bacteriocin which genes are frequently located on plasmids (Gasson, 1984, FEMS Microbiol. Letters 21, 7-10). There have been several unsuccessful attempts to use nisin resistance genes as selectable markers in conjugation and transformation of such Nis.sup.r plasmids. These attempt have failed primarily due to a high frequency of spontaneously resistant mutants. (Klaenhammer and Sanozky, 1985, J. Gen. Microbiol. 131, 1531-1541; McKay and Baldwin, 1984, Appl. Environ. Microbiol. 47, 68-74).
Froseth et al., 1988, Appl. Environ. Microbiol. 54 2136-2139 found by studying the plasmid pNP40 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3 that there was on this plasmid a close linkage between the nis.sup.r gene and a replication function. A 7.6 kb fragment of this plasmid which was designated pFM011 was found to replicate independently when transformed to Lactococcus cells. However, the nis.sup.r gene which was located on a 2.6 kb fragment was not suitable as a selectable marker since it did not allow direct or primary selection of transformants. The authors concluded that the Nis.sup.r phenotype can be used as a secondary selectable marker in cloning experiments in which direct selection is first made by using another characteristic. In their studies, this was demonstrated in co-transformation experiments by using Ery.sup.r (resistance to the pharmaceutical antibiotic erythromycin) of a shuttle vector which i.e. replicates in E. coli as the primary selectable marker.
Recently, the same authors have published the results of further experiments aiming at utilizing the Nis.sup.r phenotype of pFM011 as a selectable marker in transformation experiments in a Lactococcus sp. (J. Dairy Sci. 72, July 1989, Supplement 1, 115). However, selection of transformants by using the nis.sup.r gene as the sole selectable marker was only successful when a two-step procedure was applied comprising as a first step the plating of the transformation mixture onto M17-glucose agar having a pH at about 7.0 and containing either 0.25M sodium succinate or 30 international units (iu) nisin/ml, as for protoplast transformation or electroporation, respectively. After 3-5 days of incubation, a second step was carried out involving replica plating of presumptive transformants onto M17-glucose agar having a pH of about 7.0 and containing 0.1% Tween 20 and 20-40 iu nisin/ml and subsequent further incubation.
Such a two-step procedure is unlikely to be effective due to the step of replica plating which involves a considerably risk of missing the few transformant cells present on the first incubation medium. In case the first incubation medium has incorporated nisin it is furthermore likely that the activity hereof is decreased significantly during the long incubation period due to a low degree of stability of that bacteriocin at pH-values above 5-6. An other serious disadvantage is that the two-step procedure is time- and labour-consuming.
The present inventors have now succeeded in developing a recombinant plasmid which is highly suitable as a cloning vector in lactic acid bacteria for food starter cultures. The vector according to the invention fulfills all the requirements of a safe food-grade cloning vehicle as defined above and in the construction of the vector all theoretical potential risks associated with the use of gene technology in the improvement of food starter cultures have been taken into account and importantly, the cloning vector according to the invention comprises a carefully selected nis.sup.r gene conferring to the transformed lactic acid bacteria resistance at a very high level whereby it has now been made possible to use a nis.sup.r gene as a primary selectable marker in a direct one-step selection procedure.